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Metabolic Engineering 42 (2017) 19–24

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Metabolic Engineering

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Enhancing erythritol productivity in Yarrowia lipolytica using metabolic MARK engineering

Frédéric Carlya, Marie Vandermiesb, Samuel Telekb, Sébastien Steelsb, Stéphane Thomasc, ⁎ Jean-Marc Nicaudc, Patrick Fickersb, a Unité de Biotechnologies et Bioprocédés, Université Libre de Bruxelles, Belgium b Microbial Processes and Interactions, TERRA Teaching and Research Centre, University of Liège - Gembloux Agro-Bio Tech, Belgium c Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France

ARTICLE INFO ABSTRACT

Keywords: Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol with sweetening properties that is used by the Yarrowia lipolytica agrofood industry as a food additive. In this study, we demonstrated that metabolic engineering can be used to Erythritol improve the production of erythritol from glycerol in the yeast Yarrowia lipolytica. The best results were obtained Metabolic engineering using a mutant that overexpressed GUT1 and TKL1, which encode a glycerol kinase and a transketolase, Glycerol kinase respectively, and in which EYK1, which encodes erythrulose kinase, was disrupted; the latter enzyme is involved Transketolase in an early step of erythritol catabolism. In this strain, erythritol productivity was 75% higher than in the wild Erythrulose kinase type; furthermore, the culturing time needed to achieve maximum concentration was reduced by 40%. An additional advantage is that the strain was unable to consume the erythritol it had created, further increasing the process's eﬃciency. The erythritol productivity values we obtained here are among the highest reported thus far.

1. Introduction as mannitol, and organic acids which renders the downstream proces- sing more challenging. Recently, Yarrowia lipolytica has also been found Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol with to be an efficient erythritol producer (Rymowicz et al., 2008; Yang sweetening properties that is used by the agrofood industry as a food et al., 2014; Park et al., 2016; Mirończuk et al., 2016). additive (E968). Erythritol is noncariogenic and has been determined to Y. lipolytica is a non-conventional model yeast species that is well- be safe for human consumption even when high doses are consumed known for its unusual metabolic properties (Fickers et al., 2005; daily (Bernt et al., 1996; Kawanabe et al., 1992). It has extremely low Nicaud, 2012). Because it can secrete large amounts of proteins and digestibility and does not modify blood insulin levels (Munro et al., metabolites of biotechnological interest, Y. lipolytica has several 1998). Erythritol is widespread in nature and has been found in industrial applications, including heterologous protein synthesis and seaweed, fungi, fruit, and fermented food, although always at low citric acid production, and has been accorded GRAS status (Fickers levels (Moon et al., 2010). It is also produced by osmophilic micro- et al., 2005; Zinjarde, 2014). Y. lipolytica is highly proficient at organisms in response to osmotic stress (Hallsworth and Magan, 1997). producing erythritol and can use raw glycerol instead of glucose as its Although erythritol can be produced chemically from dialdehyde main carbon source (Rymowicz et al., 2008; Almeida et al., 2012). Raw starch, this process has never been industrialized due to its low glycerol is a byproduct of the biodiesel production or fat industries (i.e. efficiency. Instead, erythritol is most commonly generated from glucose fat saponification, stearin synthesis) and is thus available in large via fermentation processes using osmophilic yeasts (Moon et al., 2010) quantities at lower price than glucose. Moreover, glycerol allows higher namely Aurobasidium sp. (Ishizuka et al., 1989), Trigonopsis variabilis production yields as compared to glucose, making the erythritol (Kim et al., 1997), Torula sp. (Lee et al., 2000), Candida magnoliae (Ryu production process more profitable. (Rymowicz et al., 2008; et al., 2000), Pseudozyma tsubakaensis (Jeya et al., 2009), and Moniliela Tomaszewska et al., 2012; Rywińska et al., 2013). The synthesis of sp. (Lin et al., 2010). Despite the some of these processes have been erythritol from glycerol is not a redox-balanced reaction, as it requires a developed at industrial scale, they suffer from the high cost of the net amount of oxidized cofactors. However it is more advantageous fermentation media and the production of unwanted byproducts such than using glucose since synthesis of erythritol from the latter consumes

⁎ Correspondence to: Microbial Processes and Interactions, TERRA Teaching and Research Centre, University of Liège - Gembloux AgroBio Tech, Passage des Déportés, 2, 5030 Gembloux, Belgium. E-mail address: pﬁ[email protected] (P. Fickers). http://dx.doi.org/10.1016/j.ymben.2017.05.002 Received 1 March 2017; Received in revised form 2 May 2017; Accepted 19 May 2017 Available online 22 May 2017 1096-7176/ © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. F. Carly et al. Metabolic Engineering 42 (2017) 19–24

2. Materials and methods

2.1. Strains, media, and culture conditions

The Escherichia coli and Y. lipolytica strains used in this study are listed in Supplementary table 1. The E. coli strains were grown at 37 °C in Luria-Bertani medium supplemented with kanamycin sulfate (50 mg/L; Sigma-Aldrich). The Y. lipolytica strains were grown at 28 °C in YNB medium supplemented to meet the requirements of auxothrophs (Barth and Gaillardin, 1996); EG medium (glycerol 50 g/ Fig. 1. Pathway used by Y. lipolytica to synthesize erythritol from glycerol. DHAP: L, yeast extract 5 g/L and peptone 5 g/L); EPF medium (glycerol 100 g/ dihydroxyacetone phosphate; GA3P: glyceraldehyde-3-phosphate; GK: glycerol kinase; L, yeast extract 1 g/L, NH4Cl 4.5 g/L, CuSO4 0.7 mg/L, MnSO4·H2O GDH: glycerol-3P dehydrogenase; TIM: triosephosphate isomerase; TK: transketolase; ff E4PP: erythrose-4P phosphatase; ER: erythrose reductase; and EK: erythrulose kinase. 32 mg/L, and 0.72 M phosphate bu er pH 4.3) or EPB medium Dotted arrows represent the hypothetic catabolism pathway of erythrulose-P according to (glycerol 150 g/L, NH4Cl 2 g/L, KH2PO4 0.2 g/L, MgSO4·7H2O 1 g/L, Paradowska and Nitka (2009). NaCl 25 g/L and yeast extract 1 g/L). For solid media, agar (15 g/L) was added. Shake-flask cultures were performed in triplicate for eight days reduced cofactors that must be replenished through glucose oxidation. in a rotary shaker at 28 °C and 190 RPM. After 72 h of preculturing in When erythritol is synthesized from glycerol, the latter is first 35 mL of EG medium, cells were transferred to a 250 mL-flask contain- phosphorylated by a glycerol kinase (GK) before subsequently being ing 35 mL of EPF medium; initial optical density at 600 nm (OD600) was dehydrogenated by a glycerol-3P-dehydrogenase (GDH), giving rise to 0.4. Bioreactor cultures were performed in duplicate in a 2-L Biostat B- dihydroxyacetone phosphate (DHAP) (Fig. 1). DHAP is then converted Twin fermentor (Sartorius) containing 1 L of EPB medium and kept at by a triosephosphate isomerase (TIM) into glyceraldehyde-3-phosphate, 28 °C. Stirrer speed was set to 800 RPM, and the aeration rate was 1 L/ which enters into the pentose phosphate pathway, where a transketo- min. The pH was automatically maintained at 3.0 by the addition of lase (TK) converts it into erythrose-4-phosphate. The latter is depho- 20% (w/v) NaOH or 40% (w/v) H3PO4. sphorylated by an erythrose-4P phosphatase (E4PP) and reduced by an erythrose reductase (ER) to become erythritol. Depending on experi- 2.2. Quantifying biomass, glycerol concentration, and erythritol mental conditions, Y. lipolytica can also use erythritol as its main carbon concentration source. We have recently highlighted that this catabolic pathway in Y. lipolytica is similar to those present in other yeasts, including Lipomyces Biomass was determined gravimetrically after the cells had been starkeyi (Carly et al., submitted for publication)(Fig. 1). Furthermore, washed and dried at 105 °C for 24 h. Glycerol and erythritol concentra- we identified the gene EYK1 (YALI0F01606g), which encodes an tions were determined using an HPLC (Agilent 1200 series; Agilent erythrulose kinase (EK). In Y. lipolytica, disruption of EYK1 impairs Technologies) equipped with a refractive index detector and an Aminex growth on erythritol. HPX-87H ion exclusion column (300×7.8 mm; Bio-Rad). Elution was Although most of the genes involved in erythritol synthesis have performed using 15 mM trifluoroacetic acid as the mobile phase at a been described, little is known about their regulation at finer scales or flow rate of 0.5 mL/min and a temperature of 65 °C. about the pathway's limiting steps. To date, most studies seeking to improve erythritol production have used wild type strains or randomly 2.3. General molecular biology techniques generated mutants and have focused on optimizing the culture medium or culturing conditions (Rymowicz et al., 2008; Yang et al., 2014; Standard media and techniques were used for E. coli (Sambrook Mirończuk et al., 2015; Rakicka et al., 2016). However, some recent et al., 1989), and the media and techniques used for Y. lipolytica have research using Y. lipolytica has underscored the potential utility of been described elsewhere (Barth and Gaillardin, 1996). The restriction genetic engineering approaches. Mirończuk et al. (2016) found that the enzymes, DNA polymerases, and ligase were supplied by Thermo Fisher constitutive expression of genes encoding GK and GDH (i.e., GUT1 and Scientific. Genomic DNA from Y. lipolytica was prepared in accordance GUT2, respectively) in the Y. lipolytica A101 strain led to a significant with Querol et al. (1992). PCR was performed using the primers listed increase in glycerol uptake capacity. Overexpression of these genes in Supplementary table 2. DreamTaq DNA polymerase (Thermo Scien- resulted in significant increases in erythritol productivity (23% for tific) was used for cloning, and ExTaq DNA polymerase (Takara) was GUT1 only and 35% for both) in the mutants as compared to the wild used to verify genomic structure. The PCR fragments were purified from type strain; in contrast, overexpression of GUT2 alone led to a 28% the agarose gels using a GeneJet Gel Extraction Kit (Thermo Scientific). decrease in erythritol productivity. Other enzymes merit some attention DNA sequencing was performed by GATC Biotech (https://www.gatc- as well. Transketolase (TK) has been described as a key enzyme in biotech.com), and the primers were synthetized by Eurogentec (https:// erythritol synthesis in Trichosporonoides megachiliensis (Sawada et al., secure.eurogentec.com/). 2009) similarly to erythrose reductase (ER) in Candida magnolia (Ghezelbash et al., 2014). Furthermore, using proteomics, Yang et al. 2.4. Strain construction (2015) highlighted the importance of triose-phosphate isomerase (TIM) in erythritol synthesis in Y. lipolytica. Details on the genes that were overexpressed in Y. lipolytica are In this study, we sought to identify additional limits acting on provided in Table 1. The Y. lipolytica genes (namely, GUT1, GUT2, TPI1, erythritol production and thus further enhance this process. To this end, TKL1) were amplified from the genomic DNA of JMY2900. Gene YidA we constructed a set of strains that overexpressed genes encoding for was amplified from E. coli strain BL21, and gene ALR was amplified key enzymes in the erythritol synthesis pathway. We then studied the from C. magnolia strain CBS2800. A synthetic, codon-optimized version effects of different gene combinations on erythritol synthesis. In the of ALR (ALR_CO) specifictoY. lipolytica was obtained from GeneArt most productive mutants, EYK1 was also disrupted to further increase (https://www.thermofisher.com/geneart). Codon content of gene ALR erythritol productivity. was compared to Y. lipolytica CLIB122 codon usage table using Graphical Codon Usage Analyzer CGUA (http://gcua.schoedl.de/) and rare codons (below 20% in frequency in Y. lipolytica) were replaced with the most common codons coding for the same amino acid (Supplementary Fig. 1). All the gene-amplification primers were

20 F. Carly et al. Metabolic Engineering 42 (2017) 19–24

Table 1 Table 2 Genes overexpressed in Y. lipolytica. Dynamics of erythritol production and glycerol consumption in different strains grown in EPF medium in shake flasks for eight days. Reference Name Enzyme encoded Origin Modification

Strain Overexpressed Biomass qERY (g/ qGLY (g/ YP/S (g/g)

YALI0F00484g GUT1 Glycerol kinase Y. lipolytica BamHI site genes (gDCW/L) gDCW.h) gDCW.h) removed YALI0B13970g GUT2 Glycerol-3P Y. lipolytica BglII site removed JMY2900 – 5.30 0.035 0.076 0.46 dehydrogenase (WT) YALI0F05214g TPI1 Triose-P isomerase Y. lipolytica FCY205 GUT1 4.83 0.051* 0.091* 0.56* YALI0E06479g TKL1 Transketolase Y. lipolytica Intron removed, FCY206 GUT2 4.83 0.038 0.068 0.56 ClaI site removed FCY207 TPI1 5.02 0.039 0.071 0.54 EG11195 YidA Erythrose-4P E. coli FCY208 TKL1 5.36 0.040 0.068 0.59* phosphatase FCY209 YidA 4.96 0.031 0.066 0.44 FJ550210 ALR Erythrose C. magnoliae FCY210 ALR 6.24* 0.028 0.049* 0.57* reductase FCY213 GUT1-GUT2 3.62* 0.056* 0.102* 0.54 FCY214 GUT1-TKL1 4.81 0.058* 0.095* 0.61* FCY215 GUT1-ALR 4.48 0.058* 0.094* 0.61* * * designed to introduce an AvrII site at the 3′ end and either a BamHI or a FCY216 GUT1-TPI1 4.65 0.048 0.085 0.56 BglII restriction site at the 5′ end (Supplementary table 2). Introns and The values provided are the means of three independent replicates; the standard undesirable restriction sites were removed by overlap extension PCR deviations represented less than 10% of the means. The abbreviations are as follows: and site-directed mutagenesis (Higuchi et al., 1988; see Table 1 for qERY: specific erythritol production rate; qGLY: specific glycerol consumption rate; and YP/ fi details). Amplicons were puri ed from the agarose gel and then S: erythritol/glycerol conversion yield. digested using BamHI/AvrIIorBglII/AvrII restriction enzymes. The * Significantly different from JMY2900. corresponding fragments were subsequently cloned into BamHI/AvrII digested JMP1047 or JMP2563 vectors to obtain URA3 or LEU2- Alpha value was set at 0.05. All statistical tests were performed using selectable plasmids, respectively (Supplementary table 1). The correct- GraphPad Prism 6.0.2 software. ness of the resulting constructs was verified by DNA sequencing. Strain RIY203 was constructed by disrupting the EYK1 gene in the 3. Results and discussion Po1d strain as described elsewhere (Vandermies et al., 2017, in press). EYK1 P and T fragments were amplified from Po1d genomic DNA using 3.1. Overexpression of glycerol kinase increases glycerol consumption rate primer pairs EYK1-PF/EYK1-PR and EYK1-TF/EYK1-TR, respectively. and erythritol productivity The URA3 marker was amplified from the JMP113 plasmid using the primer pair LPR-F/LPR-R. Amplicons were digested with SfiI before With the goal of enhancing erythritol productivity, we first being purified and ligated, using T4 DNA ligase. The ligation products attempted to improve glycerol consumption by overexpressing the were amplified via PCR using the primer pair EYK1-PF/EYK1-TR. They genes GUT1 (which codes for GK; YALI0F00484g) and GUT2 (which were then purified and used to transform the Po1d strain. The result codes for GDH; YALI0B13970g) either separately or simultaneously in was strain RIY147, from which the URA3 marker was popped out using the Y. lipolytica strain Po1d (Fig. 1 and Supplementary table 1). For the Cre-lox recombination system and the replicative vector pRIP132 strain FCY205 (pTEF-GUT1), the specific glycerol consumption rate

(Vandermies et al., 2017, in press); this process yielded strain RIY203. (qGLY) was 20% higher than that of the wild type strain JMY2900 Expression cassettes for the GUT1, GUT2, TPI1, TKLI, YidA, ALR, (0.091 and 0.076 g/gDCW.h respectively; Table 2). This increase is and ALR_CO genes were rescued from corresponding vectors by NotI similar to that obtained for the Y. lipolytica A101 mutant that over- digestion. They were then purified from the agarose gel and used to expressed GUT1 (Mirończuk et al., 2016). In contrast, strain FCY206 transform Y. lipolytica strains Po1d or RIY203 (Supplementary Fig. 2). (pTEF-GUT2) showed a slightly lower glycerol consumption rate as Transformants were selected using YNB medium supplemented with compared to the wild type strain (Fig. 1) even though GUT2 expression uracil or leucine, depending on the nature of their auxotrophy. The was six times greater in FCY206 than in the wild type (Supplementary correctness of the strain construction was verified by performing Fig. 3). Furthermore, strain FCY213, which overexpressed both GUT1 analytical PCR on genomic DNA; depending on the marker, primer and GUT2, demonstrated just a slight increase in specific glycerol pair URA3F/61stop or LEU2F/61stop was employed. Prototrophic consumption rate compared to strain FCY205 (pTEF-GUT1) (0.102 and stains were obtained by transforming the mutants with either the 0.091 g/gDCW.h, respectively). In Y. lipolytica strain A101, an 11% JMP1047 or JMP2563 plasmid, depending on the nature of the strain's increase in glycerol consumption was observed when both GUT1 and auxothrophy. GUT2 were coexpressed as compared to when just GUT1 was overexpressed (Mirończuk et al., 2016). For strains FCY205 (pTEF-GUT1) fi 2.5. RNA isolation and transcript quanti cation and FCY213 (pTEF-GUT1-GUT2), a similar increase of qGLY was observed (12%; Table 2). Therefore, the simultaneous overexpression Shake-flask cultures were grown in EPF medium for 24 h. Cells were of GUT1 and GUT2 seems to have a combined effect on glycerol then collected at an OD600 of 0.5 and stored at −80 °C. RNA extraction consumption under our experimental conditions. However, strain and cDNA synthesis were performed as previously described (Sassi FCY213 (pTEF-GUT1-GUT2) had a 30% lower maximum biomass as et al., 2016). Primers for RT-qPCR are listed in Supplementary table 2. compared to the wild type (3.62 and 5.3 gDCW/L, respectively; Table 2). Gene expression levels were standardized using the expression level of In FCY205 (pTEF-GUT1), the specific erythritol production rate Δ the actin gene as the reference ( CT method). For genes GUT1, GUT2, (qERY) was 45% greater than in the wild type (0.051 and 0.035 g/ ff TPI1, and TKL1, the fold di erence in gene expression between the gDCW.h, respectively), while the glycerol/erythritol conversion yield -ΔΔCT mutants and the JMY2900 strain were calculated as 2 (Livak and (YP/S) was 21% higher (0.56 and 0.46 g/g, respectively). Surprisingly, Schmittgen, 2001). Samples were analyzed in duplicate. and in contrast to the findings of Mirończuk et al. (2016), the overexpression of GUT2 (strain FCY206) had no effect on specific

2.6. Statistical analysis erythritol production rate (0.038 g/gDCW.h). In strain FCY213 (pTEF- GUT1-GUT2), there was only a slight increase (9%) in the speciﬁc Statistical signiﬁcation of results was assessed by analysis of erythritol production rate as compared to that in strain FCY205 (pTEF- variance (ANOVA) followed by Dunnett's multiple comparison tests. GUT1) (0.056 and 0.051 g/gDCW.h, respectively). Since the overexpres-

21 F. Carly et al. Metabolic Engineering 42 (2017) 19–24 sion of GUT1 led to an increase in both the specific glycerol consump- occurred in the translation of ALR mRNA in strain FCY210, a codon- tion rate and the specific erythritol production rate, an additional optimized version of ALR (ALR_CO) was designed and used to construct strain, FCY212, was constructed that contained two copies of the pTEF- strain FCY211. The latter did not show significantly improved erythritol GUT1 expression cassette. However, it did not display further improve- productivity relative to strain FCY210 (data not shown). Strain FCY209, ment in erythritol productivity (data not shown). which over expressed E4PP (YidA), had a significantly lower level of erythritol production than the wild type (30 and 35 g/L, respectively) fi 3.2. Overexpression of triose isomerase and transketolase leads to an and a slower speci c erythritol production rate (0.031 and 0.035 g/ ff increase in erythritol productivity gDCW.h, respectively). This negative e ect of YidA overexpression could be linked to the ability of the YidA gene product to hydrolyze DHAP, as Genes such as TPI1, TKL1, E4PP, and ER encode key enzymes suggested by Kuznetsova et al. (2006). involved in the pathway by which erythritol is synthesized from DHAP, the end product of glycerol catabolism (Fig. 1). They were over- 3.3. The pull and push strategy to enhance erythritol production expressed separately in the Po1d strain to assess their effects on erythritol productivity. Genes encoding TIM (TPI1; YALI0F05214g) As mentioned above, strain FCY205 (pTEF-GUT1) showed a sig- fi and TK (TKL1; YALI0E06479g) were identi ed in the Y. lipolytica nificant increase in glycerol uptake, while strain FCY208 (pTEF-TKL1) genome and used to construct strains FCY207 and FCY208, respec- displayed the highest conversion yield. Hence, to further increase tively. Unfortunately, erythrose-4P-phosphatase (E4PP) has yet to be erythritol productivity, GUT1 and TKL1 were coexpressed in strain characterized in yeast. However, Kuznetsova et al. (2006) reported that, FCY214 (pTEF-GUT1-TKL1I; Supplementary Fig. 3). In shake-flask in E. coli, a member of the haloacid dehalogenase-like hydrolase cultures, this strain performed significantly better than the wild type, — — superfamily HAD13 showed a high degree of phosphatase activity displaying a 65% increase in erythritol productivity. It combined the 6 directed toward erythrose-4-phosphate (Kcat/Km value of 10 ). There- higher glycerol uptake capacity of strains FCY205 (pTEF-GUT1, 0.095 fore, the corresponding YidA gene (EG11195) was cloned, assessed for and 0.091 g/gDCW.h, respectively) and the higher conversion yields of codon compatibility in Y. lipolytica, and used to construct strain FCY208 (pTEF-TKL1, 0.61 and 0.59 g/g, respectively). To further FCY209. We were also interested in ER. Past research has suggested expand this push and pull strategy, strains FCY215 and FCY216 were that gene JX885666 in Y. lipolytica strain DSMZ70562 encodes an ER constructed; they coexpressed GUT1-ALR and GUT1-TPI1, respectively. (Ghezelbash et al., 2014). However, a BlastN search using the gene Although these two strains performed better than wild type, their fi sequence as a query did not nd a corresponding gene in the genome of erythritol production was not significantly greater than that of strain strain Po1d. Moreover, our attempts to amplify that particular gene FCY214 (pTEF-GUT1-TKL1)(Fig. 2, Table 2). This suggests that glycerol using the primers designed by Ghezelbash and colleagues (ER1 and assimilation and the redirection of glyceraldehyde-3-phosphate towards ER2; Supplementary table 2) were unsuccessful for both Po1d and the non-oxidative part of pentose phosphate pathway are the main fi DSMZ70562 (data not shown). In contrast, we did nd a corresponding limiting steps for the synthesis of erythritol. Therefore, increasing the gene (FJ550210) in C. magnolia JH110 (Lee et al., 2010) using a BlastN rate of these reactions could contribute to increasing the erythritol fi search. Consequently, it was ampli ed from the genomic DNA of C. productivity and yield. magnolia CBS2800 and used to construct strain FCY210. Next, we investigated the behavior of strain FCY214 (pTEF-GUT1- These engineered strains were grown in EPF medium for eight days TKL1) and the wild type under bioreactor conditions. The strains were (i.e., until glycerol near-exhaustion). Strains FCY207 (pTEF-TPI1) and cultured for 96 h in EPD medium, and cell growth, glycerol consump- FCY208 (pTEF-TKL1) showed increased erythritol production com- tion, and erythritol production were monitored (Fig. 3). For strain pared to the wild type (5% and 16%, respectively; Fig. 2 and FCY214 (pTEF-GUT1-TKL1), the final erythritol concentration in the Supplementary Fig. 3). Strain FCY208 (pTEF-TKL1) had a higher culture supernatant was 79.4 g/L (Table 3). This value was 42% greater conversion yield than did strain FCY205 (pTEF-GUT1) (0.59 and than that obtained for the wild type (55.8 g/L; Table 3). Under 0.56 g/g, respectively; Table 2). However, glycerol consumption was bioreactor conditions, erythritol was produced at a high, constant rate somewhat lower in the former (0.068 g/gDCW.h) than in the wild type (0.84 g/L.h) between 24 h and the end of culture (Table 3 and Fig. 3). strain (0.076 g/gDCW.h). Strain FCY210, which overexpressed the ALR This result contrasts with those obtained for Y. lipolytica strain AJD gene taken from C. magnolia, did not have increased erythritol produc- pADUTGut1/2 (constitutive GUT1/GUT2 coexpression), whose erythri- tion (Fig. 2). To circumvent any potential problems that may have tol production differed from that of the wild type (strain A101) only during the four last hours of culture (Mirończuk et al., 2016). Here, erythritol specific production rate was similar for the bioreactor and

shake-ﬂask cultures of strain FCY214 (0.057 and 0.058 g/gDCW.h, respectively), while glycerol uptake was slightly higher in the bior-

Fig. 3. Glycerol uptake (red line) and erythritol production (blue line) for FCY214 (pTEF- GUT1-TKL1 triangles) and JMY2900 (WT; circles) grown in EPB medium in a bioreactor. Fig. 2. Erythritol production of the study strains grown in EPF medium in shake ﬂasks for (For interpretation of the references to color in this ﬁgure legend, the reader is referred to eight days. the web version of this article).

22 F. Carly et al. Metabolic Engineering 42 (2017) 19–24

Table 3 the development of an eﬃcient process for producing erythritol is the Dynamics of erythritol production and glycerol consumption for bioreactor cultures of ability of Y. lipolytica to catabolize erythritol alongside glycerol. Here, JMY2900 (WT), FCY214, and FCY218. The standard deviations represented less than 10% we also constructed a strain whose erythritol catabolism was impaired. of the means. The abbreviations are the same as in Table 2. In this mutant, erythritol productivity was increased 78% relative to the JMY2900 FCY214 FCY218 wild type and maximum concentrations were obtained in 40% less time. Moreover, we achieved these values using an inexpensive medium Erythritol production (g/L) 55.8 79.4 80.6 and without having optimized culturing conditions. The next step is to Erythritol productivity (g/L.h) 0.59 0.84 1.03 q (g/g .h) 0.046 0.057 0.073 develop a glycerol fed-batch fermentation method that can further ERY DCW ﬁ qGLY (g/gDCW.h) 0.105 0.119 0.138 increase the speci c erythritol production rate as well as yield.

YP/S (g/g) 0.44 0.48 0.53 Final biomass (gDCW/L) 12.8 14.7 14.6 Funding

F. Carly and M. Vandermies each received a fellowship from the Belgian Fund for Research Training in Industry and Agriculture (FRIA), Grant No. (FC00572).

Conﬂict of interest

The authors declare that they have no conﬂicts of interest.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ymben.2017.05.002. Fig. 4. Glycerol uptake (red line) and erythritol production (blue line) for FCY218 (pTEF- GUT1-TKL1-Δeyk1; triangles) and JMY2900 (WT; circles) grown in EPB medium in a References bioreactor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). Almeida, J.R.M., Fávaro, L.C.L., Quirino, B.F., 2012. Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste. Biotechnol. Biofuels 5, 48. eactor (0.119 vs. 0.095 g/gDCW.h). Bernt, W.O., Borzelleca, J.F., Flamm, G., Munro, I.C., 1996. Erythritol: a review of biological and toxicological studies. Regul. Toxicol. Pharmacol. 24, S191–S197. 3.4. Disruption of EYK1 in strain FCY214 further increases erythritol Barth, G., Gaillardin, C., 1996. Yarrowia lipolytica. In Nonconventional Yeasts in – productivity Biotechnology. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 313 388. Carly, F., Gamboa-Melendez, H., Vandermies, M., Damblon, C., Nicaud, J.-M., Fickers, P., 2017. Identification and characterization of EYK1, a key gene for erythritol The ability of Y. lipolytica to catabolize erythritol alongside glycerol catabolism in Yarrowia lipolytica. Appl. Microbiol. Biotechnol (submitted for negatively affects erythritol productivity and conversion yield. publication). Fickers, P., Benetti, P., Wache, Y., Marty, A., Mauersberger, S., Smit, M., Nicaud, J.-M., Recently, we discovered that the gene EYK1 (YALI0F01606g) is 2005. Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its involved in erythritol catabolism (Carly et al., submitted for publica- potential applications. FEMS Yeast Res. 5, 527–543. tion). Therefore, this gene was disrupted in a strain overexpressing Ghezelbash, G.R., Nahvi, I., Malekpour, A., 2014. Erythritol production with minimum by-product using Candida magnoliae mutant. Appl. Biochem. Microbiol. 50, 292–296. GUT1 and TKL1 (Supplementary Fig. 2). As expected, the resulting Hallsworth, J.E., Magan, N., 1997. A rapid HPLC protocol for detection of polyols and strain, FCY218, was unable to consume erythritol, especially after the trehalose. J. Microbiol. Methods 29, 7–13. glycerol source had been exhausted in the culture medium (Fig. 4). In Higuchi, R., Krummel, B., Saiki, R.K., 1988. A general method of in vitro preparation and fi those conditions, erythrulose, the byproduct of erythritol oxidation, speci c mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367. started to accumulate in the culture medium after glycerol depletion, Ishizuka, H., Wako, K., Kasumi, T., Sasaki, T., 1989. Breeding of a mutant of however in small amount (less than 3% of the produced erythritol was Aureobasidium sp. with high erythritol production. J. Ferment. Bioeng. 68, 310–314. converted after 24 h of glycerol depletion). Consequently, strain Jeya, M., Lee, K.-M., Tiwari, M.K., Kim, J.-S., Gunasekaran, P., Kim, S.-Y., Kim, I.-W., Lee, J.-K., 2009. Isolation of a novel high erythritol-producing Pseudozyma tsukubaensis FCY218 performed better than strain FCY214 (glycerol specific con- and scale-up of erythritol fermentation to industrial level. Appl. Microbiol. – sumption rate: 0.138 vs. 0.119 g/gDCW.h; erythritol specific production Biotechnol. 83, 225 231. rate: 1.03 vs. 0.84 g/L.h; and conversion yield: 0.53 vs. 0.48 g/g; Kawanabe, J., Hirasawa, M., Takeuchi, T., Oda, T., Ikeda, T., 1992. Noncariogenicity of erythritol as a substrate. Caries Res. 26, 358–362. Table 3). Moreover, the maximum concentration of erythritol was Kim, S.-Y., Lee, K.-H., Kim, J.-H., Oh, D.-K., 1997. Erythritol production by controlling obtained in 40% less time than in the wild type strain, which further osmotic pressure in Trigonopsis variabilis. Biotechnol. Lett. 19, 727–729. emphasizes the potential benefits of this system. Kuznetsova, E., Proudfoot, M., Gonzalez, C.F., Brown, G., Omelchenko, M.V., Borozan, I., Carmel, L., Wolf, Y.I., Mori, H., Savchenko, A.V., et al., 2006. Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like 4. Conclusion phosphatase family. J. Biol. Chem. 281, 36149–36161. Lee, J.-K., Ha, S.-J., Kim, S.-Y., Oh, D.-K., 2000. Increased erythritol production in Torula sp. by Mn2+ and Cu2+. Biotechnol. Lett. 22, 983–986. In the past, erythritol productivity in Y. lipolytica has largely been Lee, D.-H., Lee, Y.-J., Ryu, Y.-W., Seo, J.-H., 2010. Molecular cloning and biochemical improved by classical approaches that consisted of optimizing either characterization of a novel erythrose reductase from Candida magnoliae JH110. the culture medium or culturing conditions (Rymowicz et al., 2008, Microb. Cell Factor. 9, 43. ń Lin, S.-J., Wen, C.-Y., Wang, P.-M., Huang, J.-C., Wei, C.-L., Chang, J.-W., Chu, W.-S., Miro czuk et al., 2014, Yang et al., 2014). Recently, however, 2010. High-level production of erythritol by mutants of osmophilic Moniliella sp. Mirończuk et al. (2016) reported that metabolic engineering could Process Biochem. 45, 973–979. increase the rate of glycerol catabolism, resulting in a 35% increase in Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408. erythritol productivity. In this study, we attempted to make further Mirończuk, A.M., Furgała, J., Rakicka, M., Rymowicz, W., 2014. Enhanced production of progress by increasing the flow of carbon through the erythritol erythritol by Yarrowia lipolytica on glycerol in repeated batch cultures. J. Ind. synthesis pathway, notably in the transitions from erythrose phosphate Microbiol. Biotechnol. 41, 57–64. Mirończuk, A.M., Rakicka, M., Biegalska, A., Rymowicz, W., Dobrowolski, A., 2015. A to erythritol. Our approach resulted in a 65% increase in erythritol two-stage fermentation process of erythritol production by yeast Y. lipolytica from specific production rate relative to the wild type. A major challenge in

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molasses and glycerol. Bioresour. Technol. 198, 445–455. Rywińska, A., Juszczyk, P., Wojtatowicz, M., Robak, M., Lazar, Z., Tomaszewska, L., Mirończuk, A.M., Rzechonek, D.A., Biegalska, A., Rakicka, M., Dobrowolski, A., 2016. A Rymowicz, W., 2013. Glycerol as a promising substrate for Yarrowia lipolytica novel strain of Yarrowia lipolytica as a platform for value-added product synthesis biotechnological applications. Biomass-Bioenergy 48, 148–166. from glycerol. Biotechnol. Biofuels 9, 180–191. Sambrook, J., Fritsch, E., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual: Moon, H.-J., Jeya, M., Kim, I.-W., Lee, J.-K., 2010. Biotechnological production of Vol. 2 (S.l.: Cold Spring Harbor). erythritol and its applications. Appl. Microbiol. Biotechnol. 86, 1017–1025. Sassi, H., Delvigne, F., Kar, T., Nicaud, J.-M., Coq, A.-M.C.-L., Steels, S., Fickers, P., 2016. Munro, I., Bernt, W., Borzelleca, J., Flamm, G., Lynch, B., Kennepohl, E., Bär, E., Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant Modderman, J., 1998. Erythritol: an interpretive summary of biochemical, metabolic, protein production in the yeast Yarrowia lipolytica. Microb. Cell Factor. 15, 159–169. toxicological and clinical data. Food Chem. Toxicol. 36, 1139–1174. Sawada, K., Taki, A., Yamakawa, T., Seki, M., 2009. Key role for transketolase activity in Nicaud, J.-M., 2012. Yarrowia lipolytica. Yeast 29, 409–418. erythritol production by Trichosporonoides megachiliensis SN-G42. J. Biosci. Bioeng. Paradowska, K., Nitka, D., 2009. Puriﬁcation and characterization of erythritol 108, 385–390. dehydrogenase from Mycobacterium smegmatis. Ann. UMCS Pharm. 22, 47–55. Tomaszewska, L., Rywińska, A., Gładkowski, W., 2012. Production of erythritol and Park, Y.-C., Oh, E.J., Jo, J.-H., Jin, Y.-S., Seo, J.-H., 2016. Recent advances in biological mannitol by Yarrowia lipolytica yeast in media containing glycerol. J. Ind. Microbiol. production of sugar alcohols. Curr. Opin. Biotechnol. 37, 105–113. Biotechnol. 39, 1333–1343. Querol, A., Barrio, E., Huerta, T., Ramón, D., 1992. Molecular monitoring of wine Vandermies, M., Denies, O., Nicaud, J.M., Fickers, P., 2017. EYK encoding erythrulose fermentations conducted by active dry yeast strains. Appl. Environ. Microbiol. 58, kinase as a catabolic selectable marker for genome editing in the non-conventional 2948–2953. yeast Yarrowia lipolytica. J. Microb. Methods (in press). Rakicka, M., Rywińska, A., Cybulski, K., Rymowicz, W., 2016. Enhanced production of Yang, L.-B., Dai, X.-M., Zheng, Z.-Y., Zhu, L., Zhan, X.-B., Lin, C.-C., 2015. Proteomic erythritol and mannitol by Yarrowia lipolytica in media containing surfactants. Braz. analysis of erythritol-producing Yarrowia lipolytica from glycerol in response to J. Microbiol. 47, 417–423. osmotic pressure. J. Microbiol. Biotechnol. 7, 1059–1069. Rymowicz, W., Rywińska, A., Gładkowski, W., 2008. Simultaneous production of citric Yang, L.-B., Zhan, X.-B., Zheng, Z.-Y., Wu, J.-R., Gao, M.-J., Lin, C.-C., 2014. A novel acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1. Chem. osmotic pressure control fed-batch fermentation strategy for improvement of Pap. 62, 239–246. erythritol production by Yarrowia lipolytica from glycerol. Bioresour. Technol. 151, Ryu, Y.-W., Park, C.Y., Park, J.B., Kim, S.-Y., Seo, J.-H., 2000. Optimization of erythritol 120–127. production by Candida magnoliae in fed-batch culture. J. Ind. Microbiol. Biotechnol. Zinjarde, J., 2014. Food-related application of Yarrowia lipolytica. Food Chem. 152, 1–10. 25, 100–103.